Project Summary Glycoengineering is a clinically-validated strategy to enhance the therapeutic properties of protein and peptide drugs. This strategy involves the attachment and manipulation of carbohydrates (i.e., glycans) to improve the stability, solubility, serum half-life, and activity of these drugs. A key factor in most glycoengineering is the inclusion of terminal sialic acid residues on glycans by a process known as sialylation. Sialic acid is large and carries a negative charge which serves to improve stability, decrease aggregation, slow clearance, and impede immune response. Nearly all examples of glycoengineering require eukaryotic cell culture and/or the in vitro conjugation of glycans. Unfortunately, eukaryotic cell culture can be expensive, time consuming, and can result in inconsistent and incomplete sialylation. Although in vitro glycosylation can result in similar effects, the process is expensive, difficult, and has not been scalable to a commercial level. Glycoengineering would be greatly improved if a simple host cell such as Escherichia coli was used for production of sialylated therapeutic proteins. Glycobia specializes in genetically engineering bacteria for the bottom up glycoengineering (BUG) of therapeutic glycoproteins. Since E. coli lacks native protein glycosylation pathways of any kind, BUG can produce tailored glycan structures that can be site-specifically conjugated to target proteins. The specific hypothesis behind this proposal is that glycoengineered E. coli can produce enhanced therapeutic proteins by sialylation in a short, single fermentation. In Phase I of this project we engineered E. coli to attach humanlike, sialyl-T glycans to recombinant proteins. The sialyl-T glycan is a sialylated Thomsen- Friedenreich antigen that can be found on erythrocytes in the human body. This type of glycosylation is simply not possible in any other known expression host. We also show that bacterial glycosylation improves the in vitro stability of therapeutic proteins expressed in E. coli. We anticipate that our BUG expression platform will be capable of producing sialylated proteins in a controlled, rapid, cost-effective manner. The objective of this proposal is to synthesize and advance our first drug targets from glycoengineered E. coli into preclinical testing by: (i) expressing, purifying, and characterizing glycosylated drug candidates from E. coli and (ii) testing stability, pharmacokinetics, and immunogenicity of these drug candidates in animal models. We will compare their performance to aglycosylated and asialylated versions of these same drugs to isolate the effects of sialylation. The benchmark of success for this project is the generation of positive preclinical validation data to further advance commercialization of this technology. This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of generating cost-effective glycoengineered protein drugs for both companies and patients.